This invention relates to an integrated ‘lab-on-a-pipette’ which will provide sample-to-answer single cell genetic diagnosis for preimplantation genetic diagnosis (PGD) and other forms of single cell analysis (SCA). SCA is a quickly growing field with substantial impact in prenatal testing, cancer biopsies, diabetes, stem cell research, and our overall understanding of heterogeneity in biology. However, single cell genetic analysis is challenging, inaccurate, and in many cases impossible, due to the small amount of sample (5 pg), and difficulties in handling small sample volumes (50-100 μL). The ‘lab-on-a-pipette’ device integrates a microaspiration tip with microfluidic analysis components to conduct in-situ, real-time single cell genetic diagnosis in a single disposable device. The microaspiration tip will extract and encapsulate a cell into an ultra-low volume plug (˜300 μL). The microfluidic analysis module, which includes a microheater and embedded lyophilized reagents, will perform cell lysis, polymerase chain reaction (PCR) amplification, and real-time fluorescence analysis. This solution is novel, simple (requires no integrated valves), and builds upon well established technologies for a high likelihood of success. Microfluidic devices have been successfully used to analyze low copy biomolecules (less than 1 pg) such as DNA from a single cell. Glass micropipettes are regularly used to extract single cells. Combining the two components in a single integrated system provides an elegant solution the to the difficult problem of single cell sampling and isolation. It addresses major limitations of the current preimplantation genetic diagnosis (PGD) procedures by providing a ‘one-tube’ solution to extract and analyze a single cell for genetic mutations. This eliminates fluid transfers (which cause contamination), reduces analysis time, and reduces operator and reagent costs. Moreover, the small fluid volumes (pL) used in the system increases the effective concentration of the sample, and therefore provides better amplification and improved accuracy when measuring single gene mutations. Beyond PGD, the system provides a versatile platform tool for basic research, drug discovery, and clinical diagnosis of rare or localized cell populations.
This technology can greatly improve the accuracy and reduce the costs associated with preimplantation genetic diagnosis (PGD), which will ultimately lead to healthier embryos for at-risk parents conceiving via in vitro fertilization (1% of births per year in the USA). This technology also addresses significant research challenges in single cell analysis (SCA), which relies on microscale technologies to quantiatively analyze the genome, transciptome, and metabolome of a single cell, leading to a greater understanding of heterogenaity in biology. SCA is a young but growing field which has direct implications in cancer diagnostics, undersanding diabetes and immune disorders, stem cell research, and the discovery of new drugs.
IVF is widely used today. More than 1 million children worldwide to date and about 1% of births/year in the US, i.e. more than 45,000/year, have been conceived by IVF (Goldberg). Typically, the embryo quality is assessed using light microscopy where morphological parameters such as fragmentation, number and size of blastomeres, and the nucleus status are evaluated. PGD can be used for prenatal diagnosis of known heritable chromosomal abnormalities or gene mutations (Shulman).
PGD has been used in thousands of IVF clinical cases worldwide since its inception in 1988. In PGD one cell from an in vitro embryo is extracted and biopsied, and this can be performed at different stages. Blastomere biopsy of a 6 to 8-cell embryo is performed on day 3 of IVF and involves two steps: first mechanical (razor, laser) or chemical (enzymatic) interruption of the zona pellucida and then aspiration of a blastomere (the cell produced during cleavage of a fertilized egg) with a biopsy pipette (35 μm diameter) on a manipulator (SCIENTIFIC) (Shulman) (Handyside) while another pipette on a second manipulator is holding the embryo. The procedure typically requires two embryologists. The blastomere with a visible nucleus close to the hole is targeted (SCIENTIFIC). The blastomer does not have to be completely aspirated into the pipette. The aspirated blastomere is then examined. If it has a clear nucleus and is not lysed it is transfered in PCR tubes with 5 μL of lysis buffer for molecular genetics (SCIENTIFIC).
However, blastomere biopsy's limitations decrease the likelihood of an accurate diagnosis (Shulman) (Gleicher). Limitations include: (a) Loss of viable cells (7 to 10%) due to critical steps in cell fixation for nucleate extraction (Velilla) (Shulman). (b) Contamination by ambient DNA due to contamination from extraneous sperm attached to the zona pellucida, carry-over contamination from products of former PCR reactions, DNA in reagents, or operator DNA (Shulman) (Yap) (Thornhill) (Jeanine Cieslak-Janzen) (Rechitsky) (Carson). Contamination is more disastrous than other failures, because it could allow an affected embryo to be implanted by providing a false negative (Shulman). (c) Small amount of DNA (˜5 pg) obtained from a single cell is often not sufficient for the diagnosis using current techniques (Shulman). Nested primer PCR improves allele dropout (ADO) rates; but it has a failure rate approaching 10% with one major reason being the loss of nucleic material prior to the process (Shulman).
The “lab-on-a-pipette” device integrates a micro-syringe with a lab-on-a-chip device to conduct in-situ, real-time, immediate single cell genetic diagnosis. The device will interrupt the zona pellucida, extract a cell, and perform PCR (including encapsulation, lysing, adding PCR buffer, thermal cycling, and analysis of the lysate). Microfluidic devices have been successfully used to analyze low copy biomolecules (less than 1 pg) such as DNA in a single cell (Huang) (Marcus). Microfluidic platforms have been able to manipulate cells and have provided extremely high detection sensitivity (Huang). However, they have never been combined with micromachined pipettes in a single system for in-situ diagnosis. The device addresses major limitations of the current PGD procedures by being able to analyze small amounts of DNA, eliminating cell fixation and most sources of contamination (since the cell is transferred directly from the embryo to the device for analysis), and reducing costs (less people involved in genetic diagnosis, reduced procedures and time).
For time sensitive analysis like PGD, fast analysis rates are very important. This device can complete an analysis much faster. There is no need for instance to transfer the cells to different tubes and ship the cells to a different location for analysis. In addition, amplification can be done much faster, embedded micro-heaters can heat to 100° C. in less than 10 seconds, reducing the time that it takes to complete 30 cycles to a couple of minutes from the current 10 minutes. The smaller fluid volumes in the form of plug handled by our micropipette and microfluidic device increase the effective concentration and provide better amplification with improved results and better chances of measuring single gene mutations.
On a broader scope, the lab-on-a-pipette technology can potentially become a versatile tool for the exploding field of single cell analysis (SCA). SCA is used to study heterogeneity in cell populations and in practical situations where only low cell counts are available (forensics, cancer diagnostics, and stem cell research) (Anselmetti). SCA tools are urgently needed to quantitatively record proteomic, genomic, or metabolic markers which reveal the status of the cell. Low-copy-number biomolecules (<1000 molecules/cell) have a significant function in cell operation, including signaling and regulation of gene expression (Ying), although they are often lost in the detection process and not analyzed (Gygi). Small changes of concentration or altered modification patterns of disease-relevant low abundance components can be potentially used as markers of different stages of disease such as cancer, in diagnosis, in monitoring the growth of the tumor, and response to the therapy. Molecular alterations can be used to identify cancer, determine malignancy grade, enhance diagnosis and prognosis in cancers, and clinical response to therapy (Maruvada). SCA is also important in studying cell mutations due to environmental changes, drug screening, and provides an alternative in the event of regulatory changes such as the ban on animal testing in the European Union (EU). Understanding the molecular origin of disease allows for direct treatment and the capability to predict and prevent disease. This system can detect low concentration of biomolecules such as DNA. This provides a versatile platform tool for basic research, drug discovery, and clinical diagnosis of rare or localized cell populations.
This tool can be broadly used by the research and medical community for genetic diagnosis as a fully functioning and automated single cell genetic analysis “sample-to-answer” system. The lab-on-a-pipette fills the deficiency gap for these conditions in the prior art.